Ribozymes

ABSTRACT

Compounds having highly specific endoribonuclease activity are described. The compounds of this invention, also known as ribozymes, comprise ribonucleotides having two hybridizing regions with predetermined sequences capable of hybridizing with a target RNA, a region of defined sequence and a base paired stem region.

This application is a continuation of U.S. Ser. No. 926,148, filed Aug.5, 1992, now U.S. Pat. No. 5,254,678 which is a continuation of U.S.Ser. No. 536,625, filed Aug. 14, 1990, now abandoned.

The present invention relates to a class of synthetic RNA molecules andderivatives thereof, hereinafter referred to as ribozymes, which possesshighly specific endoribonuclease activity.

A number of naturally occurring RNA molecules such as avocardo sunblotchviroid (ASBV), the satellite RNAs of tobacco ringspot virus (sTobRV) andlucerne transient streak virus (sLTSV), undergo self-catalysed cleavage.Such cleavage appears to be an essential and unique part of the lifecycle of these and other RNAs.

Self-catalysed RNA cleavage reactions share a common requirement fordivalent metal ions and neutral or higher pH, and result in theproduction of RNA with termini possessing 5' hydroxyl and 2', 3' cyclicphosphate groups (Prody et al., Science 231: 1577-1580 (1986) andBuzayan et al., Virology 151: 186-199 (1986)). The cleavage reactionsare catalysed by the RNAs themselves, presumably as a result ofconformation bringing reactive groups into close proximity. The sites ofself-catalysed cleavage in naturally occurring RNAs are located withinhighly conserved regions of RNA secondary structure (Buzayan et al.,Proc. Natl. Acad. Sci. U.S.A. 83: 8859-8862 (1986) and Forster, A.C. andSymons, R. H. Cell 50: 9-16 (1987)).

Experiments we have carried out on the satellite RNAs of tobaccoringspot virus (sTobRV) have led to the design of novelendoribonucleases (hereinafter referred to as "ribozymes"), that is,enzymes comprised of RNA which catalyse specific cleavage of RNA targetmolecules.

The term ribozyme as used in the specification refers to moleculescomprised wholly of RNA or derivatives thereof.

The ribozymes of the present invention are distinct from the RNAendoribonuclease which occurs naturally in Tetrahymena Thermophila(known as the IVS, or L-19 IVS RNA) and which has been extensivelydescribed by Thomas Cech and collaborators (Zaug, A. J. et al, Science(1984) 224: 574-578; Zaug, A. J. and Cech, T. R., Science (1986) 231:470-475; Zaug, A. J., et al, Nature (1986) 324: 429-433; publishedInternational patent application No. WO 88/04300 by University PatentsInc.). The Cech endoribonuclease has an eight base pair active sitewhich hybridizes to a target RNA sequence whereafter cleavage of thetarget RNA takes place, with a requirement for free guanosine orguanosine derivatives. The fragments which arise from cleavage containterminal 5' phosphate and 3' hydroxyl groups. The limited number ofnucleotides available for hybridization to an RNA substrate limits theeffectiveness or efficiency of the Cech endoribonuclease as in general,oligonucleotides comprising less than twelve nucleotides hybridizepoorly to target sequences. It also appears that a number of nucleotidesin the active site of the Cech endoribonuclease may need to be conservedfor efficient endoribonuclease activity. This restricts the number ofpermutations of active site sequences which can be engineered to effecthybridization to target sequences, thereby restricting the range of RNAtarget sequences cleavable by the Cech endoribonuclease. The Cechendoribonuclease also modifies RNA by adding a free guanosine nucleotideto the 5' end of cleaved RNA.

In contrast, the ribozymes of the present invention hybridizeefficiently to a wide range of target RNA sequences, and do not modifythe cleaved target RNA.

The ribozymes of the present invention comprise a hybridizing regionwhich is complementary in nucleotide sequence to at least part of atarget RNA, and a catalytic region which is adapted to cleave the targetRNA. The hybridizing region contains 9 or more nucleotides.

In a preferred aspect the ribozymes of the present invention have ahybridizing region comprising one or more arms formed of single strandedRNA and having a sequence complementary to at least part of a targetRNA, said one or more arms being associated with a catalytic regioncapable of cleaving said target RNA; and where the hybridizing regioncomprises a single arm of RNA said arm contains at least 9 nucleotides,and where the hybridizing region comprises 2 or more arms of RNA, thesum of nucleotides in said arms is greater than 9 nucleotides.

In one embodiment of the invention, there is provided a ribozyme of theformula 1 (SEQ. I.D. No. 1): ##STR1## wherein; X represents anyribonucleotide and each X residue may be the same or different; the sumof n and n' is greater than 6 and n and n' may be the same or different;an (*) represents a base pair between complementary ribonucleotides; X'and X" represent oligoribonucleotides of complementary sequence along atleast part of their length to allow base pairing between theoligoribonucleotides, or X' and X" together form a single RNA sequencewherein at least part of said sequence comprises a stem formed by basepairing between complementary nucleotides; and

optionally, an additional nucleotide selected from one of A, G, C or Umay be inserted after ¹ A in formula (1).

Region (I) of formula (1) represents the arms or flanking sequences of aribozyme which hybridize to respective portions of a target RNAsequence. The arms may hybridize along the full length of the target RNAor part thereof. An RNA catalytic region is depicted at region (II) offormula 1. The catalytic region may contain one or more additionalnucleotides which do not adversely effect catalytic activity. Suchadditions could be readily tested for ribozyme activity without undueexperimentation following the teachings of the specification. Thecatalytic region may also form part of the hybridizing region.

The oligoribonucleotides X' and X" may comprise up to 5,000 or morenucleotides.

According to a further specific embodiment of the present inventionthere is provided a ribozyme of the formula 2 (SEQ. I.D. No. 2):##STR2## wherein; X represents any ribonucleotide and each X residue maybe the same or different;

an (*) represents a base pair between complementary ribonucleotides;

n and n' are as previously defined;

m and m' are 1 or more and may be the same or different;

B represents a bond, a base pair, a ribonucleotide, or anoligoribonucleotide containing at least 2 ribonucleotides;

and optionally, an additional nucleotide selected from any one of A, G,C or U may be inserted after ¹ A in formula (2).

The ribozymes of the present invention may be prepared by methods knownper se in the art for the synthesis of RNA molecules. (For example,according to recommended protocols of Promega, Madison, Wis., USA). Inparticular, the ribozymes of the invention may be prepared from acorresponding DNA sequence (DNA which on transcription yields aribozyme, and which may be synthesized according to methods known per sein the art for the synthesis of DNA) operably linked to an RNApolymerase promoter such as a promoter for T7 RNA polymerase or SP6 RNApolymerase. A DNA sequence corresponding to a ribozyme of the presentinvention may be ligated into a DNA transfer vector, such as plasmid orbacteriophage DNA. Where the transfer vector contains an RNA polymerasepromoter operably linked to DNA corresponding to a ribozyme, theribozyme may be conveniently produced upon incubation with an RNApolymerase. Ribozymes may, therefore, be produced in vitro by incubationof RNA polymerase with an RNA polymerase promoter operably linked to DNAcorresponding to a ribozyme, in the presence of ribonucleotides. Invivo, prokaryotic or eukaryotic cells (including mammalian and plantcells) may be transfected with an appropriate transfer vector containinggenetic material corresponding to a ribozyme in accordance with thepresent invention, operably linked to an RNA polymerase promoter suchthat the ribozyme is transcribed in the host cell. Transfer vectors maybe bacterial plasmids or viral RNA or DNA. Nucleotide sequencescorresponding to ribozymes are generally placed under the control ofstrong promoters such as, for example, the lac, SV40 late, SV40 early,metallothionin, or λ promoters. Ribozymes may be directly transcribedin-vivo from a transfer vector, or alternatively, may be transcribed aspart of a larger RNA molecule. For example, DNA corresponding toribozyme sequences may be ligated into the 3' end of a carrier gene, forexample, after a translation stop signal. Larger RNA molecules may helpto stabilize the ribozyme molecules against nuclease digestion withincells. On translation the carrier gene may give rise to a protein, whosepresence can be directly assayed, for example, by enzymatic reaction.The carrier gene may, for example, encode an enzyme.

In a further aspect of the invention, there is provided a DNA transfervector which contains a DNA sequence corresponding to a ribozymeoperably linked to a promoter to provide transcription of the ribozyme.

In one preferred method of producing a ribozyme, two syntheticoligonucleotides of complementary sequence are prepared by standardprocedures (for example, using an Applied Biosystems Model 380A DNASynthesizer (Applied Biosystems Inc., Foster City, Calif. 94404)), andhybridized together. One of the oligonucleotides encodes a desiredribozyme. The respective ends of the hybridized oligonucleotidescorrespond to different restriction enzyme sites, say EcorR1 at one endand Pst1 at the other end. After cleavage with appropriate restrictionenzymes (EcoR1 and Pst1 in the above example), the double stranded DNAfragment may be cloned into a transfer vector. Where the plasmid vectorcontains an RNA polymerase promoter upstream from the DNA sequencecorresponding to a ribozyme of the present invention. RNA transcriptscorresponding to the ribozyme can be conveniently prepared eitherin-vitro or in-vivo. Where the ribozyme is comprised of two halves heldtogether by base-pairing between complementary nucleotides, each half ofthe ribozyme may be produced according to the above methods, and thehalves incubated together to form the ribozyme.

The preferred ribozymes of the present invention cleave target RNA whichcontains the sequence X°UY, where X° is any ribonucleotide, U is uraciland Y is adenine, cytosine or uracil. X°U forms part of a base pairflanking region and Y is not base paired. Preferably, but by no meansexclusively, X° is guanidine, and X°UY is GUC or GUA. Any RNA moleculecontaining these sequences can be cleaved with the ribozymes of thepresent invention. Once the sequence of an RNA transcript containing thesequence X°UY has been determined, the arms of the ribozyme sequence canbe synthesised to be complementary to, and thus hybridizable to, the RNAon the target sequence flanking the X°UY sequence. On hybridization ofthe arms of the ribozyme to the target RNA sequence flanking the X°UYsequence, the catalytic region of the ribozyme cleaves the target RNAwithin the X°UY sequence. RNA cleavage is facilitated in the presence ofmagnesium or other divalent cation at a pH of approximately 8.0.

Accordingly, the preferred ribozymes of the present invention can beengineered to cleave any RNA whose sequence is known. The high frequencyof the residues cleaved by the ribozymes in RNA (1:64 for GUC in an RNAwith random and equal frequency of base distribution) means that anumber of potential sites for ribozyme cleavage can be confidentlypredicted in any given target RNA.

According to another aspect of the present invention there is provided amethod for the inactivation of a target RNA sequence, which comprisesreacting said target RNA sequence with a ribozyme of the presentinvention.

In-vivo, that is, within the cell or cells of an organism, a transfervector such a bacterial plasmid or viral RNA or DNA, encoding one ormore ribozymes, may be transferred into cells e.g. (Llwewllyn et al., J.Mol. Biol. (1987) 195: 115-123; Hanahan et al. J. Mol. Biol (1983) 166).Once inside the cell, the transfer vector may replicate, and betranscribed by cellular polymerases to produce ribozyme RNAs which theninactivate a desired target RNA. Alternatively, a transfer vectorcontaining one or more ribozyme sequences may be transfected into cellsor introduced into cells by way of micromanipulation techniques such asmicroinjection, such that the transfer vector or a part thereof becomesintegrated into the genome of the host cell. Transcription of theintegrated genetic material gives rise to ribozymes, which act toinactivate a desired target RNA.

The ribozymes of the present invention have extensive therapeutic andbiological applications. For example, disease causing viruses in man andanimals may be inactivated by administering to a subject infected with avirus, a ribozyme in accordance with the present invention adapted tohybridize to and cleave RNA transcripts of the virus. Such ribozymes maybe delivered by parenteral or other means of administration.Alternatively, a subject infected with a disease causing virus may beadministered a non-virulent virus such as vaccinia or adenovirus whichhas been genetically engineered to contain DNA corresponding to aribozyme operably linked to an RNA promoter, such that the ribozyme istranscribed in the cells of the host animal, transfected with theengineered virus, to effect cleavage and/or inactivation of the targetRNA transcript of the disease causing virus. The ribozymes of thepresent invention have particular application to viral diseases causedfor example, by the herpes simplex virus (HSV) or the AIDS virus (HIV).

The ribozymes of the present invention also have particular applicationto the inactivation of RNA transcripts in bacteria and other prokaryoticcells, plants and animals. In bacteria, RNA transcripts of, for example,bacteriophage, which cause bacterial cell death, may be inactivated bytransfecting a cell with a DNA transfer vector which is capable ofproducing a ribozyme in accordance with the present invention whichinactivates the phage DNA. Alternatively, the ribozyme itself may beadded to and taken up by the bacterial cell to effect cleavage of thephage RNA.

RNA transcripts in plants may be inactivated using ribozymes encoded bya transfer vector such as the Ti plasmid of Agrobacterium tumefaciens.When such vectors are transfected into a plant cell, the ribozymes areproduced under the action of RNA polymerase and may effect cleavage of aspecific target RNA sequence. Accordingly, plant viruses whose RNAsequence are known, or the RNA transcripts of plant genes, may beinactivated using ribozymes.

Endogenous gene transcripts in plants, animals or other cell types maybe inactivated using the ribozymes of the present invention.Accordingly, undesirable phenotypes of characteristics may be modulated.It may, for example, be possible using the ribozymes of the presentinvention to remove stones from fruit or treat hereditry diseases inhumans which are caused by the production of a deleterious protein, orover production of a particular protein.

The present invention will now be illustrated by way of non-limitingexample only, with reference to the following non-limiting Examples, andFigures.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows RNA self cleavage sites of wild type and mutated RNAs; andan electrophoretic profile showing self-catalysed RNA cleavage products.

(a) Summarises the conserved structures associated withnaturally-occurring RNA cleavage sites in ASBV, newt satellite DNAtranscripts and the satellite RNAs of sTobRV, LTSV, velvet tobaccomottle virus, solanum nodiflorum mottle virus and subterranean clovermottle virus. Nucleotide sequences which are conserved between thesestructures are shown, while others are represented as X. Base-pairing isrepresented by "*" and the site for RNA cleavage is arrowed (Seq. ID No.5-6).

(b) Shows the conserved nucleotide sequence associated with the cleavageof the (+) strand of sTobRV RNA. The cleavage site is arrowed (Seq IDNo. 7)

(c) An in vitro mutant of sTobRV containing an insertion of eightnucleotides (shown boxed) together with a flanking duplication of threenucleotides (UGU resides 7 to 9) is shown (Seq. ID No. 8).

(d) Sub-cloned HaeIII fragments of wild type sTobRV and the D-51 invitro mutant were each transcribed in both the (+) and (-) orientationsand radiolabelled transcripts fractionated by polyacylamide gelelectrophoresis. The positions of uncleaved 159 and 170 base transcriptsfrom the wild type (WT) and mutant (D-51) sequences are arrowed; sizesof cleavage products are shown.

FIG. 2 shows the nucleotide sequence of a ribozyme and the products ofribozyme cleavage separated by gel electrophoresis.

(a) The inserted nucleotides in the D-51 mutant (FIG. 1c) contains aBamHI restriction endonuclease site. BamHI was used to split to mutantDNA, and the two sequences were sub-cloned and transcribed separately invitro. The RNA transcripts are shown schematically, with potentialbase-pairings between the RNAs indicated "*". The fragment containingthe arrowed site for cleavage is referred to as S-RNA, the fragmentcontaining the ribozyme is designated Rz-RNA (Seq. ID No. 9-10).

(b) [³² P]-Rz-RNA (101 bases) was incubated alone (lane 1), and withunlabelled S-RNA (lane 2). [³² P]-S-RNA was incubated alone (lane 3),and with unlabelled and ³² P labelled Rz-RNAs (lanes 4 & 5respectively).

FIG. 3 shows a schematic model of a ribozyme according to one embodimentof the present invention. Region A represents the cleavage sequencewithin the target RNA. Region B represents a catalytic region, andregions C represent the arms of the ribozyme (Seq. ID No. 11-12).

FIG. 4 shows the design of ribozymes targeted against the CAT(chloramphenicol acetyl transferase) gene transcript. Ribozymes, termedRzCAT-1, 2 and 3, were targeted against three sites within an 835 basein vitro transcript of the CAT gene. The relative locations of thecleavage sites on the transcript are shown schematically with theflanking bases numbered (a). The three ribozyme sequences are shown ((b)to (d)) with their target sequences (Seq. ID No. 13-21). Amino-acidsequences of the CAT gene are numbered and the predicted sites for RNAcleavage arrowed. RzCAT-1 and 3 contain 24 base sequences derived from(+) strand sTobRV (region B, FIG. 3), while RzCAT-2 contains a singleU-A change in this region.

FIG. 5 shows the results of CAT RNA cleavage with ribozymes RzCAT-1 to3.

(a) The [³² P]-CAT RNAs were gel fractionated after incubation alone (-)or with one of the three ribozymes, RzCAT-1 to 3 (lanes 1, 2 & 3respectively). The location of full-length transcript is shown arrowed.

(b) 5' Terminal base analysis. The 3' fragments produced by ribozymecleavage of CAT mRNA were [5'-³² P]-kinased, gel purified, subjected tocomplete nuclease digestion and the released terminal residuesfractionated by pH 3.5 polyacrylamide gel electrophoresis. The 5'terminal nucleotides, determined by reference to markers (lane M), wereA, U and G for the fragments produced by RzCAT-1 to 3 (lanes 1,2 and 3respectively.

FIGS. 6a and 6b shows a time course of ribozyme (RzCAT-1) catalyticactivity against CAT RNA. The amounts of the 139 nucleotide cleavageproduct were quantified and plotted. FIG. 6b shows the accumulation ofthe 139 base fragment with time, after polyacrylamide gelelectrophoresis.

FIG. 7a-7f shows the relative rates of cleavage of CAT RNA underdifferent temperature conditions. Substrate RNA is represented by asolid line. In each case cleavage product is represented by a brokenline.

FIGS. 8a-8c depicts three ribozymes (corresponding to RzCAT-2) havingarms or flanking sequence of varying length (Seq. ID No. 22-26)

FIG. 9 depicts a scheme for producing a catalytic anti-sense RNAmolecule having multiple catalytic domains. The CAT gene sequencecontained in the (+) strand of M13 ssDNA is subcloned into pGEM 4vector. The resulting recombinant is transcribed with T7 RNA polymeraseto produce the catalytic anti-sense RNA.

FIG. 10 shows ribozymes hybridizing to target sequences containing theGUA (10a) (Seq. ID No. 29-30) and GUU (10b) (Seq. ID No. 31-32) motifsin CAT mRNA.

FIG. 11 shows sites for self catalysed RNA cleavage in citrus exocortisviroid (CEV) RNA and its complement (Seq. ID No. 33-35).

FIG. 12 shows the ribozyme RzCEV25x(+) hybridized to target CEV RNA(a),and a gel electrophoretic profile of (+)CEV RNA and complementary (-)CEV RNA incubated with RzCEV25x(+) (b, lanes 1 and 2 respectively.Cleavage product is arrowed) (Seq. ID No. 36-37).

FIG. 13 shows the ribozyme RzCAT-2 hybridizing to its target sequence(a) and ribozyme RzSCMoV (b). The catalytic domain in each ribozyme isboxed. Differences in the catalytic region of RzSCMoV, when comparedwith RzCAT-2 are marked (Seq. ID No. 38,39,41).

FIG. 14 shows the ribozyme RzCEV-2 hybridizing to a target sequence incitrus exocortis viroid (CEV) RNA. The cleavage site corresponds tonucleotide-336 in the CEV RNA sequence. The alteration in nucleotidesequence in the catalytic domain, when compared with the catalyticdomain of sTobRV, is circled (a) (Seq. ID No. 42-43). FIG. 14 (b) showsan electrophoretic profile of a control [(-) strand of CEV] RNA, lane 7and the (+) strand of CEV RNA lane 8, after incubation with RzCEV2.

FIG. 15 shows the ribozyme RzCAT-2 (a) compared with ribozyme RzCAT-2B(b). Catalytic domains are boxed. Changes is the catalytic domain ofRzCAT-2B compared with RzCAT-2 are also boxed (Seq. ID No. 38-40,44).

FIG. 16 shows a map of plasmid pJ35SN; and

FIG. 17 is a graphic presentation of the average of four experiments onthe inhibition of CAT expression in plants (tobacco protoplasts).

The following Examples are given to illustrate the present invention andare not to be construed as limiting the present invention.

Reactions and manipulations involving DNA, such as ligations,restriction enzyme digestions, bacterial transformation, DNA sequencingetc. were carried out according to standard techniques, such as thosedescribed by Maniatis et al (molecular Cloning, Cold Spring Harbour,1982). Manipulations involving RNA were also carried out according tostandard techniques, such as those described by Uhlenbeck (Nature 328,596-600 (1987)) and Haseloff and Gerlach (Nature 334, 585-591 (1988)).

EXAMPLE 1

Self-Catalyzed Cleavage of Mutated sTobRV RNA:

A concensus of the domains associated with naturally-occurring RNAcleavage sites in ASBV, newt satellite DNA transcripts and the satelliteRNAs of sTobRV, LTSV, velvet tobacco mottle virus (VMoV), solanumnodiflorum mottle virus (SNMV) and subterranean clover mottle virus(SCMoV) as shown in FIG. 1a. Nucleotide sequences which are conservedbetween these structures are shown, while non-conserved sequences arerepresented as X. An extra U is positioned after residue ¹ A in LTSV(+)strand.

The domain associated with the self-catalysed cleavage of the (+) strandof sTobRV was studied to ascertain the enzymic substrate activity withinthis domain. First, cloned sTobRV cDNAs were mutagenised using anoligonucleotide linker (BamH1) insertion protocol.

Construction of a Vector for in vitro Expression of sTobRV:

A 160 bp Taq 1-Spe 1 fragment of sTobRV cDNA was isolated from pSP653(Gerlach et al. 1985, Virology 151: 172-185) and ligated to Acc 1-Spe 1cut, phosphatase-treated pGEM 4 to reform the Acc 1 site. A resultingclone was linearized with Acc 1, phosphatase-treated and a 359 bp Taq 1fragment of the sTobRV cDNA was inserted. The resulting clones werescreened for the presence of a circularly permuted 520 bp sTobRV cDNAsequence containing the terminally redundant residues 277 to 81 (pTTS).The sTobRv sequence is flanked by promoters for T7 and SP6 RNApolymerases, and in vitro transcription gave rise to RNAs of (+) or (-)orientation which contained two sites for self-cleavage.

In vitro Mutagenesis:

The plasmid pTTS (50 ug) was linearized with BamH 1, treated with S1nuclease and religated, to remove a unique BamH 1 site. The resultingconstruction, pTTS-B, was treated with 2×10⁻⁴ units DNase 1 in 20 mMTris-HCl pH 7.0, 15 mM MnCl₂ for 10 mins. at 37° C. The resulting linearDNAs were trimmed and/or end-filled using T4 DNA Polymerase, andpurified by 0.7% LGT agarose gel electrophoresis and extraction. KinasedBamH 1 linker sequences (CGGATCCG) were ligated to the linearizedplasmid overnight at room temperature in the presence of 5% polyethyleneglycol. Subsequently, the reactions were BamH 1 digested, and the linearplasmid DNAs repurified by 0.7% LGT agarose gel electrophoresis (thiswas found necessary to remove last traces of circular plasmid, togetherwith unligated linkers). Plasmids were recircularized using T4 DNAligase and transformed into E. coli DH-1. Colonies (greater than 1000)were scraped from agar plates, grown in liquid culture to saturation anda mixed population of plasmid DNAs prepared. The mixed sTobRV cDNAinserts were excised by restriction enzyme digestion at flanking EcoR1and Pst1 sites, purified by 1% LGT agarose gel electrophoresis, andsub-cloned into EcoR1-Pst1 cut, phosphates-treated pGEM 4. The resultingtransformants were again pooled, grown in liquid culture and plasmid DNAprepared. The plasmid DNAs were treated with BamH1, to cleave only thoseplasmids containing a BamH1 linker sequence, and the linear forms wereagain purified by two rounds of 0.7% LGT agarose gel electrophoresis,recircularized with T4 DNA ligase, and transformed into E. coli DH-1.Individual transformants were screened for the approximate position ofthe inserted BamH1 linker within the sTobRV sequence by restrictionenzyme digestion, sub-cloned into M13 mp19 and sequenced via thedideoxynucleotide chain termination technique.

A library of sTobRV mutants resulted, and nucleotide sequence analysisshowed that each mutant contained an inserted BamH1 linker sequence(CGGATCCG) together with flanking duplicated or deleted sTobRVsequences. The mutants were transcribed in vitro and the RNAs assayedfor their ability to undergo cleavage. From these experiments, a52-nucleotide sequence was identified as containing both the substrateand cleavage portions of sTobRV RNA. This 52-nucleotide sequence,depicted in FIG. 1b, contained the domain of conserved sequence requiredfor self-cleavage of other RNAs (FIG. 1a). One mutant, designated D-51,contained an eight nucleotide BamH1 linker sequence inserted betweenthree duplicated sTobRV nucleotides numbered 7 to 9. This mutantunderwent self-catalysed RNA cleavage.

97 and 108 base-pair HaeIII fragments containing the 52-nucleotidecleavage sequence of the wild type and D-51 RNAs (as shown in FIGS. 1band 1c) were excised from sequenced plasmid clones. The fragments wereligated into the Smal site of pGEM4 and screened to obtain bothorientations of the insert. The plasmids were linearised using EcoR1 and(+) and (-) strand RNAs of lengths 159 and 170 bases were transcribedusing 200 units/ml T7 RNA polymerase in 50 mM Tris-HCl, pH 7.5, 10 mMNaCl, 6 mM MgCl₂, 2 mM spermidine, 1000 units/ml RNasin, 500 μM ATP, CTPand GTP with 200 μM [α³² P] UTP. RNAs were fractionated byelectrophoresis on a 10% polyacrylamide, 7 molar urea, 25% formamidegel, and autoradiographed.

As shown in FIG. 1d, no cleavage of the (-) strand RNA transcripts wasobserved. This was as expected, as the (-) strand did not contain aself-catalysed cleavage site. With the (+) strands of both the wild typeand D-51 sequences, cleavage took place, with cleavage of the D-51 RNAbeing somewhat less efficient than that of the wild type (FIG. 1d). Thisexperiment indicates that the single stranded loop region at theright-hand side of the 52-nucleotide sequence involved in theself-catalysed cleavage of RNA is non essential.

Separation of Enzymic and Substrate Activities:

Using the BamH1 restriction endonuclease site inserted into D-51, theflanking HaeIII-BamH1 and BamH1-HaeIII fragments were obtained and eachwas sub-cloned into an E. coli plasmid suitable for in-vitrotranscription. This led to the elimination of the mutatedsingle-stranded loop from the self-cleavage domain, splitting the regioninto two RNA segments (FIG. 2a). The smaller HaeIII-BamH1 fragmentcontained nucleotides 321 to 9, including the actual site of cleavageand was termed the S fragment. The BamH1-HaeIII fragment containingsTobRV nucleotides 7 to 48 was termed the ribozyme or Rz fragment. TheE. Coli plasmids used for in-vitro transcription were pGEM3 and pGEM4(Promega, Madison, Wis., U.S.A.). these expression plasmids contain:

(a) an origin of replication;

(b) selectable drug resistance (Amp^(r)) gene;

(c) a multiple cloning site flanked by RNA polymerase promoters whichcan be used for in vitro production of transcripts.

T7 DNA polymerase treated, Kpn1 digested Rz-pGEM3 and Xbal digestedS-pGEM4 were transcribed using SP6 RNA polymerase under the sameconditions as set out above.

As shown in FIG. 2, both the S and Rz-RNAs showed no significantdegradation when incubated alone (FIGS. 2b, lanes 1 and 3) underconditions suitable for highly efficient self-cleavage (50° C., 20 mMMgCl₂, pH 8.0). The labelled Rz-RNA also appeared unaltered afterincubation with the S-RNA (FIG. 2b, lanes 2 and 5). However, when theS-RNA was mixed with the Rz-RNA, efficient cleavage of the S-RNAoccurred (FIG. 2b, lanes 4 and 5) producing two fragments. The productsizes were consistent with cleavage of the S-RNA (84 bases) at thenormal site between nucleotides #359 and #1, to give 5' and 3' proximalfragments of 67 and 17 nucleotides, respectively. This shows that theS-RNA acted as a substrate for ribonucleolytic cleavage by the Rz-RNA,which acted in a catalytic fashion.

A model of a ribozyme based on the catalytic region of sTobRV RNA isshown in FIG. 3. The ribozyme has two arms or flanking sequences ofsingle stranded RNA shown at C, hybridizing to complementary sequenceson a substrate RNA, i.e. RNA to be cleaved. Each flanking sequence shownat C, contains 8 ribonucleotides. The number of nucleotides contained inregion C is not critical. Sufficient nucleotides must, however, bepresent to allow the ribozyme to hybridize to a target RNA. Fournucleotides in each region C appears to be the minimum number forhybridization.

The catalytic region B contains sequences which are highly conserved innaturally-occurring cleavage domains (see FIG. 1a). From a comparisonwith cleavage domains of the known sequences, the length of the basepair stem II is unimportant, as is the presence of an associated loop atone end thereof.

The cleavage site within the target RNA is depicted at A (in FIG. 3) asGUC. On the basis of our experiments (not shown), and others by Koizumi(FEBS LETT 288; 228-230 (1988); and FEBS LETT 239; 285-288 (1988)) onthe cleavage sites in naturally occurring RNAs, the sequences GUA, GUC,CUC, AUC and UUC also act as cleavage sites within RNA.

EXAMPLE 2

Demonstration of the Design, Synthesis and Activity of Ribozymes withNew and Highly Specific Endoribonuclease Activity:

As an illustration of this invention, three ribozymes have beendesigned, which are targeted against the transcript of a commonly usedindicator gene derived from bacteria, Tn9 Chloramphenicol AcetylTransferase (CAT), which can provide antibiotic resistance in bacteria,plants and animals and can be easily assayed. These ribozymes,designated RzCAT-1 to 3 correspond to potential GUC cleavage sites inCAT RNA at positions 139-140, 494-495 and 662-663 respectively. Thesequences of these ribozymes are depicted in FIG. 4. In each case, theflanking sequences of the ribozyme which hybridize to the target CATRNA, were 8 nucleotides in length. The catalytic region was chosen tocorrespond to that of sTobV RNA, shown in FIG. 3.

The CAT gene was obtained from pCM4 and sub-cloned as a BamH1 fragmentinto pGEM-32 (from Promega, Madison, Wis., U.S.A.). This plasmid waslinearised with HindIII and CAT gene transcripts were obtained using T7RNA polymerase with 220 uM [α-³² P]UTP. Ribozyme sequences weresynthesised as oligodeoxynucleotides, Rz CAT-1, 2 and 3, respectively.They were kinased, ligated with phosphatased treated, EcoRI-PstI cutpGEM4 and incubated with the Klenow fragment of DNA polymerase 1 beforebacterial transformation. EcoRI linearised plasmids were transcribedwith T7 RNA polymerase to produce ribozyme RNAs. Ribozymes wereincubated with CAT transcript in 50 mM Tris-HCl pH 8.0, 20 mM MgCl₂ at50° C. for 60 min, and the products fractionated by 5% polyacrylamide 7Murea, 25% formamide gel electrophoresis prior to autoradiography.

When the 840-nucleotide CAT transcript was incubated with any one of thethree ribozymes, efficient and highly sequence-specific cleavageoccurred (FIG. 5) producing 2 RNA fragments in each reaction. Thefragment sizes were consistent with the predicted sites for cleavage(i.e. 139 and 696, 494 and 341, 662 and 173 base fragments were the 5'and 3' products from RzCAT-1 to 3 catalysed cleavage respectively). Theconditions required for these ribozyme-catalysed cleavages were similarto those observed for naturally occuring cleavage reactions (Foster, A.C. and Symons, R. H., Cell 49: 211-220 (1987) and Foster, A. C. andSymons, R. H. Cell 50: 9-16 (1987)), with more efficient cleavageoccuring at elevated pH, temperature and divalent cation concentrations(data not shown). When present in molar excess, the three ribozymescatalysed almost complete cleavage of the CAT RNA substrate after 60min. in 50 mM Tris HCl, pH 8.0, 20 mM MgCl₂ at 50° C. Under similarconditions with 0.1 μM substrate and 3 μM ribozymes, the T_(1/2) of CATmRNA substrate was 3.5, 3.5 and 2.5 min. in the presence of RzCAT-1 to 3respectively. The ribozyme sequences were inactive against thecomplement of the substrate RNA (i.e. the (+) strand), and in the formof oligodeoxyribonucleotides (data not shown). The 3' terminal cleavagefragments from each ribozyme catalysed reaction were isolated and 5' ³²P-kinased (50 mM TrisHCl pH 9, 10 mM MgCl₂, 10 mM DTT with 50 uCi γ-³² PATP and 5 units T4 polynucleotide kinase for 30 min. at 37° C.).Efficient kinasing of the fragments indicated that they possessed 5'terminal hydroxy groups, similar to those produced in naturally occuringcleavage reactions.

The terminal nucleotide of the fragments produced by cleavage of the CATsequences by RzCAT-1 to 3 were determined. Briefly, radiolabelledfragments were purified on a 5% polyacrylamide gel and digested with anequal volume of 500 units/ml RNase T1, 25 units/ml RNase T2 and 0.125mg/ml RNaseA in 50 mM ammonium acetate pH 4.5 for 120 min. at 37° C. Theproducts were fractionated on a 20% polyacrylamide gel containing 25 mMsodium citrate, pH 3.5 and 7 molar urea. FIG. 5b shows that the cleavageof the CAT sequences by RzCAT-1 to 3 occurs precisely before nucleotidesA, U and G respectively.

The terminal sequence of the CAT gene fragments were determined directlyusing the partial enzymatic digestion technique (Donis-Keller et al.,Nucleic Acids Res. 4: 2527-2538 (1980)), using base-specific partialribonucleolytic cleavage. The sequence of the fragments confirmed thatcleavage occurred at the expected locations within CAT RNA (not shown).

Enzymatic Catalysis:

To demonstrate that ribozymes cause cleavage of the CAT mRNA substratein a catalytic manner, each was incubated with a molar excess ofsubstrate, under conditions which should favour both efficient cleavageand product dissociation. FIGS. 6a and 6b show the results of anexperiment where after 75 min. at 50° C., pH 8.0 in 20 mM MgCl₂, 10pmoles of RzCAT-1 had catalysed specific cleavage of 163 pmoles of atruncated CAT mRNA (173 bases) substrate to give 5' and 3' fragments of139 and 34 bases, respectively. On average, each ribozyme hadparticipated in greater than ten cleavage events. After 75 minutes at50° C. some non-specific cleavage of RNA was noticed due to the extremeconditions, but 70% of the remaining intact RNAs (163 pmoles) hadaccumulated as the 139 base fragment. Similar results were obtained forRzCAT-2 and 3 (data not shown), and thus each acts as an RNA enzyme.

EXAMPLE 3

The Effect of Temperature on Ribozyme Activity:

The effect of reaction temperature on the in-vitro rate of ribozymeactivity was examined.

A time course of reactions for ribozymes RzCAT-1 to 3 on CAT RNAsubstrate at 37° C. and 50° C. was carried out.

In this experiment, reactions for each ribozyme were set up induplicate, using reaction conditions for ribozyme cleavage set out inExample 2. One reaction was incubated at 37° C., the other at 50° C.Samples were removed at time points to 90 minutes and the extent ofreaction was analysed by denaturing polyacrylamide gel electrophoresis.FIGS. 7a-7f shows the time course of reaction for each of the ribozymesRzCAT-1 to 3 at 37° C. and 50° C. The reaction rate of each ribozymeincreases with increased reaction temperature.

The time taken for 50% (t_(1/2)) cleavage of CAT RNA is set out in Table1.

                  TABLE 1    ______________________________________                               RzCAT-3    RzCAT-1          RzCAT-2   t1/2 (mins.)    ______________________________________    50° C.            3.5          3.5       2.5    37° C.            55.0         70.0      65.0    ______________________________________

As shown in Table 1, the rate of reaction of ribozymes at 37° C. isapproximately 20 times slower than the rate of reaction at 50° C.

EXAMPLE 4

The Effects of Varying Arm Lengths of Ribozymes (or Flanking Sequence)on Ribozyme Catalytic Activity:

The arms or flanking sequences of a ribozyme (region (I) of formula 1)hybridize the ribozyme to a target RNA whereafter cleavage of the RNAtakes place. In this experiment, the effect on cleavage rate of a targetsequence by altering the extent of complementarity and subsequent lengthof base pairing of the ribozyme arms to the target sequence wasinvestigated.

Ribozymes were produced with 4, 8 and 12 base complementarity to thetarget sequence RzCAT-2 on each arm (FIGS. 8a-8c). The ribozymes wereprepared according to the methods of Example 2. Ribozyme activity wasdetermined by incubating the ribozyme RNA with CAT RNA as describedpreviously.

The ribozyme having a 4 base complementarity on each arm did not cleavethe substrate RNA. The ribozyme with 8 base complementarity on each armcleaved the CAT substrate as did the ribozyme having 12 basecomplementarity. Ribozymes with 12 base complementarity cleaved targetRNA more efficiently, as judged by reaction rate in-vitro than didribozymes having a lesser number of base complementarities. Even thoughit appears necessary to have more than four base complementarity,increasing the length of the hybridizing region of ribozymes increasestheir reaction rate.

In a second experiment, the reaction efficiency of a ribozyme having (a)complementarity to the entire length of the CAT transcript target RNAand (b) multiple catalytic domains, was investigated.

Four GUC target sites in CAT RNA sequences were chosen. Ribozymecatalytic domains against these sites were "inserted" into a completeanti-sense (-) sequence for the CAT transcript and catalytic activitytested.

The four sites chosen were the three specified by RzCAT-1 to 3 describedpreviously, and a further site which may be represented as follows (Seq.ID No. 3-4). ##STR3## where "192" refers to amino acid 192 in the CATpolypeptide and an refers to the site of cleavage.

Oligodeoxyribonucleotides containing ribozyme catalytic domains andspanning each of these cleavage sites were used for M13 mutagenesisexperiments to produce a sequence containing the entire complement ofthe CAT sequence but with the four ribozyme catalytic domains insertedwithin it. M13 mutagenesis was performed by binding of oligonucleotidescontaining ribozyme insertions to single stranded M13 DNAs containinguracil, followed by synthesis of complementary DNAs containing theinsertion. The complementary DNAs were recovered following cloning in anappropriate Escherichia coli strain, (T. A. Kunkel 1985, Proc. Natl.Acad. Sci. U.S.A. 82: 488-492). The resultant double-stranded cDNA wascloned in an in-vitro expression vector to produce ribozyme RNA usingthe T7 polymerase transcription system. Ribozyme activity was determinedby incubation of ribozyme RNA with CAT transcript followed by gelelectrophoresis of the reaction mixture, after glyoxal treatment todenature nucleic acids.

Autolytic cleavage occurred at all the expected sites on the CATtranscript. Accordingly, the flanking sequences of arms or a ribozymemay extend along the full length of the RNA transcript which is to becleaved.

FIG. 9 schematically shows the production of catalytic anti-sense RNAcontaining each of the four ribozymes. The catalytic anti-sense RNAcontains approximately 900 bases.

Under the above reaction conditions, the ribozyme and target sequencesform high molecular weight complexes presumably by extensive basepairing. A strong denaturing treatment such as glyoxal treatment isrequired to resolve reaction products during electrophoresis.

EXAMPLE 5

Target Sequences for Ribozyme Cleavage:

The GUA motif in mRNA was tested to see whether a ribozyme would effectRNA cleavage at this sequence.

A specific site in CAT mRNA, including the GUA motif (FIG. 10a) waschosen and an appropriate ribozyme sequence was prepared and tested foractivity. The ribozyme contained arms of 8 ribonucleotides.

Synthetic oligonucleotides corresponding to the ribozyme of FIG. 10 wereprepared according to Example 2 and double-stranded cDNA cloned into anin-vitro expression vector (pGEM 4, see above) in E. coli in order toproduce ribozyme RNA using the T7 polymerase transcription system.Ribozyme activity was determined by incubation of ribozyme RNA with CATmRNA, followed by gel electrophoresis of the reaction mixture aspreviously described.

The ribozyme effected cleavage at the GUA target site (not shown).Accordingly, the motif GUA in RNA is a substrate for ribozymes of thepresent invention. This was not completely unexpected since onenaturally occuring cleavage site in the satellite RNA of lucernetransient streak virus requires recognition of a GUA site.

Similarly, a GUU motif in the CAT RNA target sequence was tested with anappropriate ribozyme (See FIG. 10b), and cleavage was effected.

EXAMPLE 6

Ribozyme Cleavage of Viral RNA:

Viroid RNA, in the form of citrus exocortis viroid RNA, was cleavedusing a ribozyme of the present invention.

Two GUC target sites were chosen in citrus exocortis viroid (CEV) RNA.One site in the complementary strand sequence was also chosen. Ribozymeswere prepared against all of these sites and tested for activity. Theribozymes were designated CEV9x(+), CEV9x(-) and CEV25x(+). FIG. 11shows the three cleavage sites in CEV RNA for each of these ribozymes.

Ribozymes were prepared according to previous methods. RibozymeRzCEV25x(+) is shown in FIG. 12. This ribozyme cleaves the GUC motif atnucleotide 116 of CEV RNA.

FIG. 12(b) shows cleavage of CEV RNA with ribozyme RzCEV9x(+). Nocleavage is observed with ribozyme RzCEV9x(-).

This experiment indicates that ribozymes are active against target RNAsequences from diverse sources. This is to be expected, as all RNAs areformed from the basic ribonucleotide building blocks of adenine,guanine, cytosine and uracil, regardless of their animal, plant ormicrobial origin.

EXAMPLE 7

Examples of Ribozymes Having Variable Catalytic Domains

A ribozyme targeted against a CAT-2 site was prepared using thecatalytic domain sequence from the satellite RNA of subterranean clovermottle virus (SCMoV). Twelve base complementarity of ribozyme armflanking sequence was incorporated into the design of the ribozymeRzSCMoV. The ribozymes RzCAT-2 and RzSCMoV are shown at FIGS. 13a and13b respectively. The loop region of RzSCMoV contains 5 nucleotides,having the sequence AAAUC. This is to be contrasted with the loop regionof RzCAT-2 which contains 4 nucleotides having the sequence AGAG. Inaddition, RzSCMoV contains a C in the catalytic region, in place of U*in RzCAT-2. The different sequences in RzSCMoV when compared withRzCAT-2 are marked.

The RzSCMoV was produced according to Example 2. RzSCMoV was active,yielding two cleavage products as expected.

In another experiment, the citrus exocortis viroid (CEV) target site atnucleotide -336 in its complementary RNA was cleaved using a ribozyme(RzCEV2) having the sequence set out in FIG. 14a. The loop regiondesignated with the letter "L" in FIG. 14 comprises six nucleotideshaving the sequence 3'-CCTATA-5'. This is distinct from the loop regionof sTobRV which comprises four nucleotides having the sequence3'-AGAG-5'. This ribozyme cleaves target CEV complementary RNA atposition -336 as shown in the electrophoretic profile of FIG. 14b.

This experiment indicates that the number of nucleotides, and nucleotidesequence of the loop region is unimportant in ribozyme activity. Inthese experiments, the ribozyme was produced according to methodspreviously described in the specification.

In another experiment, the effect of base pairing in the catalyticdomain (stem region) on ribozyme activity was investigated.

A modified ribozyme corresponding to RzCAT-2 but containing four extrabase pairs was prepared and tested. In FIG. 15a, the sequence of theribozyme Rz CAT-2 is shown hybridized to target CAT RNA. The testribozyme is shown at 15b, with the additional base pairs boxed. The testribozyme had comparable activity to that of RzCAT-2. This indicates thatthe base paired region of the ribozyme catalytic domain may be ofvariant length, without effecting catalytic activity.

We have observed (data not shown) that the stable in vivo form of sTobRVRNA transcripts expressed in transgenic plants is primarily circular,presumably due to ligation of 5' and 3' termini. Therefore, the use oftwo autolytic cleavage sites flanking a sequence of interest in an invivo RNA transcript is likely to lead to a circularized product whichmay have greater stability than linear transcripts. This approachappears to provide a novel method for in vivo stabilization of ribozymesequences. This is termed circularization.

EXAMPLE 8

In-vivo Activity of Ribozymes:

The in-vivo activity of ribozymes in plant cells is investigated in thisExample.

Experimental Protocol:

Plasmids containing anti-CAT (CAT═chloramphenicol acetyl transferase) orcombined anti-CAT/ribozyme gene constructions (see below) wereintroduced into tobacco protoplasts in the same amount and proportionrelative to each other, along with another plasmid which contained afunctional CAT gene construction. CAT activities were measured andcompared with the base level of gene activity.

Materials and Methods:

(a) Electroporations and CAT assays.

These were performed as described in Llewellyn et al. J. Mol. Biol.(1987) 195:115-123. Briefly, protoplasts of Nicotiana plumbaginifolialine T5 were prepared from a suspension two days after subculture,suspended in 10 mM HEPES, pH 7.2, 150 mM NaCl, 0.2M mannitol andadjusted to a density of 3×10⁶ /ml. Electroporation was carried outusing a single 50 ms pulse at 250 V. Protoplasts were diluted 10-foldand cultured and for 20 hr. at 26° C. in the dark. They were disruptedby sonication and extracts obtained. The extracts were normalized forprotein content and assayed for CAT activity in vitro using ¹⁴C-chloramphenicol and acetyl CoA. Reaction products were separated bythin layer chromatography and visualized by autoradiography. Extent ofreactions were calculated by the production of radioactive productderivatives from the ¹⁴ C-chloramphenicol template.

(b) Gene Constructions.

Gene constructions were introduced into 0.1 ml protoplast suspensions asplasmid DNAs which had been purified from bacteria by extraction and twocycles of CsCl equilibrium density gradient centrifugation. They wereresuspended in 10 mM Tris/1 mM EDTA/pH=7.5 for use.

The active CAT gene construction was borne on the plasmid designatedpCAT7+. It was derived by fusion of a CAT gene sequence (from plasmidpCM4, see T. J. Close and R. Rodriguez, 1982, Gene 20:305-316) into theplasmid pJ35SN (derived from p35SN, W. L. Gerlach et al., 1987, Nature328:802-805) so that the active gene construction was: ##STR4## 35Srefers to the 35S CaMV (cauliflower mosiac virus) promoter, NOS tonopaline synthetase polyadenylation signal, T/C to transcription.

Along with 0.2 ug of pCAT7+ there were added various gene constructionsin excess as described below. The gene constructions were containedwithin plasmids with the following designations:

pJ35SN--This vector plasmid, a map of which is shown in FIG. 16,contains a 35S CaMV promoter and plant nopaline synthase 3'polyadenylation signal, which may be depicted as follows: ##STR5##pCAT7---This contains the CAT gene sequence inserted into pJ35SN suchthat transcription will result in the production of the antisense CATRNA, which may be depicted as follows: ##STR6## pCAT19---This containsthe CAT gene with four catalytic ribozyme domains included within it,(see Example 4 and FIG. 9), inserted into pJ35SN such that transcriptionwill result in the production of antisense CAT RNA containing fourcatalytic ribozyme domains, which may be depicted as follows: ##STR7##Results:

The following table shows the relative CAT activities in cells 20 hoursafter electroporation. Activity is expressed as per cent conversion ofchloramphenicol substrate in a 1 hour assay.

    ______________________________________    Treat-          ug Plasmid Electroporated                                   %    ment  pCAT7+   pJ35SN   pCAT7- pCAT19- Conversion    ______________________________________    1A                                      0    1B                                      0    2A    0.2      18                      21    2B    0.2      18                      46    3A    0.2      9         9             28    3B    0.2      9         9             32    4A    0.2               18             26    4B    0.2               18             19    5A    0.2      9                9      19    5B    0.2                       9      22    6A    0.2                      18      14    6B    0.2                      18      16    ______________________________________     (for each treatment "A" and "B" are duplicates)

The following conclusion can be drawn from these results:

(a) The introduction of the CAT gene construction results in significantCAT activity--compare 2A, B with 1A, B. There is variability betweenduplicates. From the trends seen in the other samples (see "b" and "c"below) it is likely that 2A shows an abnormally low activity.

(b) Concomitant introduction of an antisense gene construction resultsin a decrease in the level of activity--compare 3A, B and 4A, B with 2B.The extent of the decrease is related directly to the level of theantisense gene added as plasmid--compare 3A, B and 4A, B.

(c) Concomitant introduction of the combined antisense/ribozyme geneconstruction results in a decrease in gene activity--compare 5A, B and6A, B with 2B. Furthermore, the decrease is more marked than for thecorresponding levels of antisense gene constructions--compare 5A,B with3A,B, and 6A,B with 4A,B.

The average results for four in-vivo experiments are shown in FIG. 17.In this FIGURE, "control" represents treatment 2. "Antisense" representstreatment 4. "Catalytic" represents treatment 6 and "Background"represents treatment 1.

The catalytic ribozyme inhibits CAT activity an average of 47%, comparedto an average of 34% for an antisense ribozyme.

The introduction of ribozyme-bearing genes into plant cells inhibits theactivity of genes against which they are targeted. Furthermore, theinhibition is greater than for corresponding antisense RNA molecules.

These results show that ribozymes will be active in animal, plant ormicrobial cells against a range of target RNA molecules.

The mechanisms of action of the ribozymes in this Example is unclear.For example, the antisense ribozyme may irreversibly hybridize to atarget RNA and catalyse phosphodiester bond cleave at one or moreselected target sites along the target RNA. Alternatively, cellularenzymes may unwind the antisense RNA from its target sequence, such thatthe target RNA is cleaved into two or more fragments.

EXAMPLE 9

In-vivo Activity of Ribozymes in Animal Cells:

The activity of ribozymes in inactivating a target RNA in mammaliancells in demonstrated in this Example.

Materials and Methods:

The active gene constructions encoding ribozymes were transfected intothe widely available monkey kidney cell line COS1 by electroporation. Inthis method, 3×10⁶ /ml COS1 cells suspended in a buffered saline with10% FCS (foetal calf serum), were contacted with various gene constructsand an electric discharge applied to effect electroporation of DNA intothe cells. The transfected cells were incubated at 37° C. for 48 hoursin culture medium before assay for CAT and luciferase activity.

CAT gene constructs were borne on the plasmid designated pTK CAT(Miksicek et al., Cell 46: 283-290, 1986). This plasmid was derived bythe introduction of a CAT gene sequence into the plasmid pSV2 such thatit is under the control of the thymidine kinase promoter of the herpessimplex virus.

Gene constructs encoding ribozymes were borne on the plasmid pSV232A (DeWet et al., Molecular and Cellular Biology 7: 725-737, 1987) containingthe luciferase gene fused to the SV40 early promoter. DNA encodingribozymes was ligated into the XbaI site at the 3' end of the luciferasegene according to the standard methods of Maniatis et al. (MolecularCloning, A Laboratory Manual, Cold Spring Harbour, 1982).

The following constructs were prepared using standard techniques ofManiatis et al. (Supra): pFC58--This plasmid vector contains DNAencoding ribozyme RzCAT-1 fused to the 3' end of the luciferase gene ina non-functional orientation.

This may be depicted as follows: ##STR8## where 232A refers to pSV232Asequences, SV40 early refers to the early promoter of SV40 and small Tis DNA encoding the small T intervening sequence of SV40. This constructresults in production of an RNA molecule encoding luciferase and theribozyme RzCAT-1, the latter being in an orientation such that it wouldnot be expected to be catalytic.

pFC4--This plasmid is the same as pFC58 except that RzCAT-1 is replacedwith RzCAT-3.

pFC1-6--This plasmid is the same as pFC58 except that RzCAT-1 isreplaced with RzCAT-3 in the sense orientation (5'-3').

pFC20--This plasmid is the same as pFC1-6 except that RzCAT-3 isreplaced with RzCAT-2 having eight nucleotide flanking sequences.

pFC12--This plasmid is the same as pFC20 except that the ribozymeRzCAT-2 contains twelve nucleotide flanking sequences.

pFC50--This plasmid contains the CAT gene with four catalytic ribozymedomains included within it (see Example 4 and FIG. 9) in the senseorientation (5'-3'), which on transcription gives rise to an inactiveribozyme. This plasmid may be depicted as follows: ##STR9## pFC54--Thisplasmid is the same as pFC50, except that the CAT gene and the ribozymedomains are in the antisense (3'-5') active orientation.

pFC64--This plasmid shares the SV40 promoter and polyadenylation signalswith pFC50 and contains the wild type CAT gene with no inserted ribozymedomains. This gene is in an antisense orientation, and thus does notproduce CAT protein.

pFC65--This plasmid is the same as pFC64 except that the wild type CATgene is in the sense (5'-3') orientation and is thus productive of CATprotein.

Assays:

Luciferase activity was assayed according to the methods of De Wet etal. (Supra). Briefly, COS cells were lysed 48 hours after transfection,and the cell lysate incubated with luciferin, the substrate ofluciferase, and luminesence detected using a scintillation counter.

CAT activity was also measured using COS cell lysates (cell lysates weredivided into two, and each portion assayed either for luciferase or CATactivity), according to the method of Sleigh, M.J., Anal. Biochem. 156:251-256, (1986).

In the in-vivo assays, pFC58 and pFC4 did not effect CAT activity intransfected cells. This activity was designated 100% CAT activity and 0%CAT suppression. CAT activity in cells transfected with other plasmidswas measured relative to pFC58. The % of CAT suppression was measured as##EQU1## normalised to luciferase production. CAT_(test) =CAT assayresult for test constructs. CAT control=CAT assay for control constructs(pFC4 and pFC58).

Luciferase production is an internal control for electroporation, andgives a measure of ribozyme production within each individuallyelectroporated tissue culture plate.

Results:

    ______________________________________    Experiment (i)                                    % CAT    Treat-          μg plasmid electroporated/1.5 × 10.sup.6                                    Suppres-    ment  pTKCAT    pFC58   pFC20 pFC1-6                                        pFC12 sion    ______________________________________    1     5                 2                 56    2     5                 1     1           53    3     5                             2     40    4     5         2                          0    ______________________________________     All treatments were carried out in duplicate and an average value given.

    ______________________________________    Experiment (ii)                                      %                                      CAT                                      Sup-    Treat- μg plasmid electroporated/1.5 × 10.sup.6                                      pres-    ment   pTKCAT    pFC-1-6  pFC20 pFC12 pFC4  sion    ______________________________________    5      5         2                          75    6      5                  2                 75    7      5                  4                 62    8      5         1        1                 70    9      5                        4           51    10     5                              2      0    ______________________________________     Treatments 5 to 10 were carried out in duplicate and an average value     given.

    ______________________________________    Experiment (iii)           μg plasmid electroporated/           1.5 × 10.sup.6 cells                             % CAT    Treatment             pTKCAT    pFC1-6    pFC4  Suppression    ______________________________________    11       5         2               66    12       5                   2      0    ______________________________________     Treatments were carried out in quintuplicate and an average value given.

    ______________________________________    Experiment (iv)                                    % CAT    Treat-          μg plasmid electrophorated                                    Suppres-    ment  pTKCAT    pFC50   pFC54 pFC64 pFC65 sion    ______________________________________    13    5         2                         0    14    5                 2                 26    15    5                       2           2    16    0                             2     NA    ______________________________________     Each of treatments 13 to 16 were carried out in quadruplicate.     The sense CAT construction (treatment 16) was productive of high levels o     CAT activity in its own right. Therefore % suppression is not applicant     (NA).

A number of experiments were also conducted with the TK promoter ofpTKCAT replaced with the human metallothionein promoter. When this CATconstruct was co-transfected into COS1 cells with plasmids encoding oneor more ribozymes, a marked decrease in CAT activity was observed.

The above results clearly demonstrate the in-vivo inactivating activityof ribozymes in animal cells.

Whilst the effectiveness of the ribozymes in vivo, is believed to becaused by one or more catalytic regions which are capable of cleavage ofa target RNA, the presence of such regions in "Antisense" RNA typeribozymes may not actually lead to cleavage in vivo if the entireRNA/antisense RNA molecule does not fall apart. However, regardless ofwhether the molecule falls apart or not, the foregoing Examplesdemonstrate the effectiveness of the ribozyme in inactivating the targetRNA. Thus, the invention is applicable to all ribozymes having acatalytic region capable of causing cleavage and a hybridizing region,regardless as to whether cleavage actually occurs in the target RNA.i.e. The hybridizing region may be so large as to cause the combinedRNA/ribozyme to stay together and prevent the target RNA being cleavedinto separated components even though the catalytic region is itselfcapable of causing cleavage.

    __________________________________________________________________________       SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii ) NUMBER OF SEQUENCES: 44    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 11 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    NNCUGAN GAGN       11    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 25 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (ix) FEATURE:     (A) NAME/KEY: misc.sub.-- feature     (B) LOCATION: 15     (D) OTHER INFORMATION: /note=" N represents a bond, a base     pair, a ribonucleotide, or an      oligonucleotide containing at least 2      ribonucleotides"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    NNCUGAN GAGNNNNN NNNNCGAA AN     25    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    HisHis AlaVal CysAsp Gly      5    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CAUCAUG CCGUCUGU GAUGGC      21    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 15 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    NNNNCUG ANGAGNNN       15    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 10 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    NNNCGAA ACN       10    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 52 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    CCUGUCA CCGGAUGU GUUUUCCG GUCUGAUG AGUCCGUGA GGACGAAA CAGG  52    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 63 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CCUGUCA CCGGAUGU CGGAUCCG UGUGUUUU CCGGUCUGA UGAGUCCG UGAGGACG AAAC 60    AGG        63    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 17 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CCUGUCA CCGGAUGU CG      17    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 46 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GAUCCGU GUGUUUUC CGGUCUGA UGAGUCCG UGAGGACGA AACAGG   46    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    NNNNNNN NNNNNNGU CNNNNNNN NNNNNNN     30    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    NNNNNNN NCUGAUGA GUCCGUGA GGACGAAA CNNNNNN    38    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    AlaPhe GlnSer ValAla Gln      5    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GCAUUUC AGUCAGUU GCUCAA      21    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GAGCAAC UCUGAUGA GUCCGUGA GGACGAAA CUGAAAU    38    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 6 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    PhePhe ValSer AlaAsn      5    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    UGUUUUU CGUCUCAG CCAAUC      21    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    UUGGCUG ACUGAUGA GUCCGAGA GGACGAAA CGAAAAA    38    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 6 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    PheHis ValGly ArgMet      5    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GCUUCCA UGUCGGCA GAAUGC      21    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    AUUCUGC CCUGAUGA GUCCGUGA GGACGAAA CAUGGAA    38    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 14 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    IleGlu AsnMet PhePhe ValSer AlaAsn ProTrp ValSer      5  10    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 42 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    AUUGAGA AUAUGUUU UUCGUCUC AGCCAAUC CCUGGGUGA GU   42    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    CUGACUG AUGAGUCC GAGAGGAC GAAACGA     30    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    UUGGCUG ACUGAUGA GUCCGAGA GGACGAAA CGAAAAA    38    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 47 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    AGGGAUU GGCUGACU GAUGAGUC CGAGAGGA CGAAACGAA AAACAUA   47    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 67 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    NNNNNNN CUGANGAG NNNNNNNN NNCGAANN NNNNNNCUG ANGAGNNN NNNNNNNC GAAA 60    CNNNNNN        67    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    NNNNNNN NCUGANGA GNNNNNNN NNNCGAAA CNNNNNN    38    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 17 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    AAGACCG UAAAGAAA AA      17    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    UUUUUCU UCUGAUGA GUCCGUGA GGACGAAA CGGUCUU    38    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 17 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    GAUAGUG UUCACCCU UG      17    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    CAAGGGU GCUGAUGA GUCCGUGA GGACGAAA CACUAUC    38    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 12 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    GAAGUCC UUCAG       12    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 12 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    AGGGUCA GGUGA       12    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 12 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    GAAGUCG AGGUC       12    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 42 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    UCCCCGG GGAAACCU GGAGGAAG UCGAGGUC GGGGACAGC UG   42    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 63 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    CAGCUGU CCCCGACC UCCUGAUG AGUCCGUG AGGACGAAA CUUGCUCC AGGUUUCC CCGG 60    GGA        63    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 10 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    AsnMet PhePhe ValSer AlaAsn ProTrp      5  10    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    AAUAUGU UUUUCGUC UCAGCCAA UCCCUGG     30    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 47 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    AGGGAUU GGCUGACU GAUGAGUC CGAGAGGA CGAAACGAA AAACAUA   47    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 48 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    AGGGAUU GGCUGACU GACGAGUC CCUAAAGG ACGAAACGA AAAACAUA   48    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    CCUGCAG GGUCAGGU GAGCAG      21    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 45 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    CUGCUCA CCUCUGAU GAGUCCGA TATCCGGA CGAAACCCU GCAGG   45    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 55 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: RNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    AGGGAUU GGCUGACU GAUGAGUC CGUCCGAG AGGACGGAC GAAACGAA AAACAUA  55

We claim:
 1. A compound having the formula: ##STR10## wherein each Xrepresents a ribonucleotide which may be the same or different;whereineach of (X)_(n) and (X)_(n') represents an oligoribonucleotide having apredetermined sequence which is (a) capable of hybridizing with an RNAtarget sequence to be cleaved and (b) does not naturally occurcovalently bound to the sequences A--A--A--G--C-- and X--C--U--G--A--,respectively, such RNA target sequence not being present within thecompound; wherein each of n and n' represents an integer which definesthe number of ribonucleotides in the oligonucleotide with the provisothat the sum of n +n' is greater than or equal to 14; wherein each *represents base pairing between the ribonucleotides located on eitherside thereof; wherein each solid line represents a chemical linkageproviding covalent bonds between the ribonucleotides located on eitherside thereof; wherein a represents an integer which defines a number ofribonucleotides with the proviso that a may be 0 or 1 and if 0, the Alocated 5' of (X)_(a) is bonded to the G located 3' of (X)_(a) ; whereineach of m and m' represents an integer which is greater than or equal to1; wherein each of the dashed lines independently represents either achemical linkage providing covalent bonds between the ribonucleotideslocated on either side thereof or the absence of any such chemicallinkage; and wherein (X)_(b) represents an oligoribonucleotide which maybe present or absent with the proviso that b represents an integer whichis greater than or equal to 2 if (X)_(b) is present.
 2. A compound ofclaim 1, wherein each of n and n' is greater than or equal to
 6. 3. Atransfer vector comprised of RNA or DNA or a combination thereofcontaining a nucleotide sequence which on transcription gives rise to acompound of any of claims 1 and
 2. 4. A transfer vector according toclaim 3 which is a bacterial plasmid or phage DNA.
 5. A method of usingthe transfer vector of claim 3 to produce the compound in a host cellwhich comprises: introducing the transfer vector into a suitable hostcell and culturing the host cell under conditions to express thetransfer vector so as to thereby produce the compound in the host cell.6. A method of using the transfer vector of claim 3 which comprisesintroducing a suitable vector into a suitable eukaryotic host cell andculturing the host cell so as to effect transcription of the nucleotidesequence contained within the transfer vector and thereby produce thecompound encoded by such nucleotide sequence, which compound is capableof hybridizing with and specifically cleaving an RNA target sequenceswithin the eukaryotic host cell.